Origins of Water Molecules in the Photosystem II Crystal Structure

May 23, 2017 - The cyanobacterial photosystem II (PSII) crystal structure includes more than 1300 water molecules in each monomer unit; however, their...
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Origins of Water Molecules in the Photosystem II Crystal Structure Naoki Sakashita,† Hiroshi C. Watanabe,†,‡ Takuya Ikeda,† Keisuke Saito,†,‡ and Hiroshi Ishikita*,†,‡ †

Department of Applied Chemistry, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

‡

S Supporting Information *

ABSTRACT: The cyanobacterial photosystem II (PSII) crystal structure includes more than 1300 water molecules in each monomer unit; however, their precise roles in water oxidation are unclear. To understand the origins of water molecules in the PSII crystal structure, the accessibility of bulk water molecules to channel inner spaces in PSII was investigated using the water-removed PSII structure and molecular dynamics (MD) simulations. The inner space of the channel that proceeds toward the D1-Glu65/D2-Glu312 pair (E65/E312 channel) was entirely filled with water molecules from the bulk region. In the same channel, a diamond-shaped cluster of water molecules formed near redoxactive TyrZ in MD simulations. Reorientation of the D2Leu352 side chain resulted in formation of a hexagonal water network at the Cl−2 binding site. Water molecules could not enter the main region of the O4−water chain, which proceeds from the O4 site of the Mn4CaO5 cluster. However, in the O4−water chain, the two water binding sites that are most distant from the protein bulk surface were occupied by water molecules that approached along the E65/E312 channel, one of which formed an H-bond with the O4 site. These findings provide key insights into the significance of the channel ends, which may utilize water molecules during the PSII photocycle.

I

cluster (O4−water chain18). The PSII crystal structure shows several characteristic water molecules near the Mn4CaO5 cluster. A water molecule at W53914 (W56719) forms a significantly short H-bond with the O4 site of the Mn4CaO5 cluster (OW539−O4 bond length, 2.50 Å14 or 2.32 Å19), which led Suga et al. to propose the possible involvement of the W539 and O4 moiety in the mechanism of water oxidation.19 Both O4 and W539 are involved in the proton-conducting O4− water chain.17,18,20 A diamond-shaped cluster of water molecules21 comprised of four water molecules is present near TyrZ. The diamondshaped cluster donates an H-bond to the phenolic O of TyrZ, lowers the pKa of TyrZ to that of the Nε site of D1-His190, and is responsible for formation of a significantly short, low-barrier H-bond between TyrZ and D1-His190 (OTyrZ−NεD1‑His190 bond length, 2.46 Å14).21 The diamond-shaped cluster is also involved in the H-bond network of the E65/E312 channel, which proceeds toward the protein bulk surface via the Cl−1 moiety.16 Cl−1 seems likely to be required to progress through the S2 to S322,23 and S3 to S0 transitions,24 as previously described.16 On the other hand, a role of the second chloride, Cl−2, identified in the PSII crystal structure14,15 is unclear. Cl−2

n photosystem II (PSII), the Mn4CaO5 cluster catalyzes the water-splitting reaction, 2H2O → O2 + 4H+ + 4e− (reviewed in refs 1−3), which is an essential process for photosynthesis and bioenergetics. Protons are released in response to changes in the oxidation state (the Sn state, where n represents the number of oxidation steps) of the oxygen-evolving complex; this occurs with a typical stoichiometry of 1:0:1:2 for the S0 → S1 → S2 → S3 → S0 transitions, respectively. Potential proton transfer pathways and water intake channels for these transitions have been reviewed (e.g., refs 4−11). The slow-exchanging water molecule (Wslow) and the fastexchanging water molecule (Wfast) are candidate substrates in the Mn4CaO5 cluster.12 In the S0 to S1 transition, the exchange rate of Wslow decreases significantly by a factor of 600, suggesting that deprotonation of Wslow occurs. Wslow may be a μ-OH bridge of the Mn4CaO5 cluster in S0.13 Wfast is assumed to release the proton in the S2 to S3 transition. It is unclear whether these water molecules are among the 1300 water molecules identified in each monomer unit of the cyanobacterial PSII crystal structure.14,15 The PSII crystal structure shows three major channel spaces from the Mn4CaO5 moiety toward the protein bulk surface,7,9 i.e., the channel that proceeds toward the protein bulk surface via the Cl−1 binding moiety and the D1-Glu65/D2-Glu312 pair (E65/E312 channel16), the water chain that proceeds from the O1 site of the Mn4CaO5 cluster (O1−water chain17), and the water chain that proceeds from the O4 site of the Mn4CaO5 © XXXX American Chemical Society

Received: March 10, 2017 Revised: April 27, 2017 Published: May 23, 2017 A

DOI: 10.1021/acs.biochem.7b00220 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry and five water molecules form a hexagonal network in the crystal structure.14,15 D1-Asn298 accepts an H-bond partner of the Nδ site of D1His190 and simultaneously donates an H-bond with a chain of water molecules. The water chain near D1-Asn298 was proposed to serve as a possible proton transfer pathway from TyrZ.3,14,25 These water molecules identified in the crystal PSII structure can be considered to be either exchangeable (exchangeable water molecule) or less exchangeable (less exchangeable water molecule) with bulk water. Less exchangeable water molecules are more likely to be incorporated into the protein interior of the PSII crystal structure during the assembly of the protein complex. In terms of the continuity of the photocycle, substrate water molecules must be at least exchangeable water molecules that can approach the catalytic site in the PSII interior from the bulk region when the previous substrate molecules are consumed. To understand the origins of water molecules in the PSII crystal structure, here we investigated the accessibility of bulk water molecules to the channel inner spaces of the PSII protein using the water-removed PSII structure and molecular dynamics (MD) simulations. Positions of the water molecules obtained using the MD simulations were compared with those in the crystal structure. The water molecules reproduced by the MD simulations are considered to enter from the outside of the protein complex and are thus designated as exchangeable water molecules, whereas those not reproduced by the MD simulations are considered to be incorporated into the protein interior during assembly of the protein complex and are designated as less exchangeable water molecules. In the protein interior, less exchangeable water molecules may also be involved in proton transfer pathways (e.g., the narrow pore of the proton-conducting O4−water chain17), because the activation energy of proton transfer is the lowest when all the water molecules are strongly H-bonded in the H-bond network.26 Exchangeable water molecules may also be involved in water intake channels (e.g., aquaporin). The analysis presented here will provide insights into the connectivity of the catalytic Mn4CaO5 site with the protein bulk surface and a key to identifying possible substrate water molecules, which approach the catalytic site in the PSII interior from the bulk region during the PSII photocycle.

V=

∑ Kb(b − beq)2 + ∑ Kθ(θ − θeq)2 bond

+

∑ dihedral

+

qiqj ⎤ ⎥ εR ij ⎥⎦

angle

Vn [1 + cos(nϕ − γ )] + 2

⎡ A B ⎢ ij − ij 12 ⎢ R ij 6 nonbond ⎣ R ij

∑

(1)

where Kb, Kθ, and Vn are bond, angle, and dihedral parameters, respectively, and beq, θeq, and γ are equilibrium values, n is the periodicity of dihedral angles, Aij and Bij are the Lennard-Jones parameters, q is the atomic charge, and ε is the dielectric constant. For standard amino acid residues, we employed the AMBER ff14SB force field parameter set.28 For cofactors and the lipids, we employed the generalized Amber force field (GAFF) parameter set,29 the GLYCAM06 force field parameter set,30 and the LIPID11 parameter set.31 For ligation of histidine to the Mg atom in chlorophyll a, we used the parameters for bacteriochlorophyll a.32 For heme, the parameters were taken from the AMBER parameter database [Giammona, D. A. (1984) Ph.D. Thesis, University of California, Davis, CA (http://research.bmh.manchester.ac.uk/bryce/amber/)]. For the non-heme Fe cluster, we obtained the parameters using the MCPB.py program33 (see Supporting Information Data Set 1 for the parameters). The atomic charges of most cofactors, including TyrZ, were determined by fitting the electrostatic potential in the neighborhood of these molecules, by using the RESP procedure34 (Table S2). After geometry optimization, the electronic wave functions were calculated by the restricted density functional theory (DFT) method with the B3LYP functional and 6-31G* basis sets, using the Gaussian 09 program35 for chlorophyll a, pheophytin a, plastoquinone, βcarotene, heptyl-1-thiohexopyranoside, digalactosyl diacyl glycerol (DGDG), 1,2-distearoyl-monogalactosyl-diglyceride, and TyrZ, and by the unrestricted DFT method with the B3LYP functional and LACVP** basis sets, using the JAGUAR program36 for the high-spin non-heme Fe cluster and the low-spin hemes. The atomic charges of 1,2-dipalmitoylphosphatidylglycerol and sulfoquinovosyldiacylglycerol (SQDG) were determined as electrostatic potential (ESP) charges; the electronic wave functions were calculated by the restricted DFT method with the B3LYP functional and 6-31G** basis sets, using JAGUAR.36 The atomic charges of the Mn4CaO5 cluster, including the ligand groups, were determined as electrostatic potential (ESP) charges (Table S2); the electronic wave functions were calculated by the unrestricted DFT method with the B3LYP functional and LACVP** basis sets, using JAGUAR.36 The cluster was considered to be in the S1 state with ferromagnetically coupled Mn atoms; the total spin S = 7, and the resulting Mn oxidation state (Mn1, Mn2, Mn3, Mn4) = (III, IV, IV, III). O1−O5 were treated as unprotonated O2−. O5 is suggested to be deprotonated13,37−40 or protonated15,41 in S1. Because O5 has no direct H-bond partner and is not allowed to form an Hbond with water molecules in the presence of W2 and W3,18,42 the O5 protonation state is unlikely to affect this result. The four ligand water molecules, W1−W4, and the ligand amino acid groups were fixed to the ligation sites of the Mn4CaO5 cluster via classical bonds (Table S3). The parameters of the

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METHODS Molecular Model and Force Field Parameters. As a basis for the computations, the X-ray crystal structure of cyanobacterial PSII (Protein Data Bank entry 3ARC)14 was used. To analyze water permeability and distribution in the channel inner spaces, all water molecules identified in the protein interior, except for ligand water molecules W1−W4 of the Mn4CaO5 cluster, were removed from the crystal structure. Hydrogen atoms were generated and energetically optimized using CHARMM version 40b;27 the positions of all nonhydrogen atoms were fixed, and titratable groups were kept in their standard protonation states (i.e., deprotonated acidic groups, protonated basic groups, and neutral histidine residues) except for those listed in Table S1. In the D1-Glu65/D2Glu312 pair (OD1‑Glu65−OD2‑Glu312 bond length, 2.52 Å14), D2Glu312 was protonated. For MD simulations, we employed the following potential energy function B

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Figure 1. (a) Water distribution after an equilibrating MD run at 0 ns (1 snapshot), 0−5 ns (100 snapshots), 10−15 ns (100 snapshots), 25−30 ns (100 snapshots), and 45−50 ns (100 snapshots). Dotted blue lines indicate H-bonds that form the diamond-shaped cluster of water molecules near TyrZ.21 (b) Root-mean-square deviations (RMSD) of the protein backbone positions with respect to the PSII crystal structure in angstroms. (c) Comparison of the geometry near the O1−water chain between the water-removed PSII structure obtained by the 50 ns MD simulation (green) and the PSII crystal structure (cyan14). Red spheres indicate the water molecules whose positions were reproduced in the 50 ns MD simulation using the water-removed PSII structure (i.e., exchangeable water molecules), whereas cyan spheres indicate the water molecules whose positions were not reproduced (i.e., less exchangeable water molecules). The latter form the H-bond network near D1-Asn298.

and soaked in 78889 flexible water models (SPC-Fw),44 using CHARMM-GUI.45 Water molecules were absent in the resulting PSII interior. After structural optimization with positional restraints on heavy atoms of the PSII assembly, the system was heated from 0.001 to 300 K over 5.0 ps. The positional restraints on heavy atoms were gradually released over 16.5 ns. While the position restraints were being released, some water molecules gradually penetrated the protein interior. After an equilibrating MD run for 45 ns, a production run was

Mn4CaO5 cluster were adjusted exclusively to maintain the QM/MM-optimized geometry of the cluster during MD simulations, which may not ensure proper dynamics of the Mn4CaO5 cluster, including the ligand moieties (see Supporting Information Data Set 1 for the parameters). The channel space was analyzed using CAVER.43 MD Simulations. The water-removed PSII assembly described above was embedded in a lipid bilayer consisting of 546 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) C

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Biochemistry conducted over 5.0 ns for sampling of the water distribution. All equilibrating simulations were conducted using the MD engine AMBER 1446 with the SHAKE algorithm for hydrogen constraints.47 NAMD version 2.1048 was used for the production run with an MD time step of 0.5 fs without hydrogen constraints. For temperature and pressure control, the Berendsen thermostat and barostat were employed in the equilibrating process,49 while the Langevin thermostat and piston were used in the production run.50,51

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RESULTS AND DISCUSSION MD simulations indicated that the root-mean-square deviation of the protein backbone atoms in the water-removed PSII structure compared to those in the PSII crystal structure reached a plateau at ∼35 ns (Figure 1b). Below, we focus on the MD trajectory obtained after 45 ns. In the resulting geometry, some side chains, in particular H-bond partners of water molecules in the original PSII crystal structure, were slightly displaced, e.g., in the region near the O1−water chain (Figure 1c); however, no significant conformational changes (e.g., backbone conformational changes) were observed. The presence or absence of water molecules in the geometry obtained in the 50 ns MD simulation seems to be strongly associated with water permeability and exchangeability in the local geometry of PSII. It was found that most of the water molecules found in the crystal structure were reproduced by the MD simulations (designated as exchangeable water molecules), whereas some were not reproduced (designated as less exchangeable water molecules). Among 103 water molecules in the protein interior near the Mn4CaO5 cluster of the PSII crystal structure, 90 water molecules could be attributed to either exchangeable water molecules or less exchangeable water molecules, comparing the positions and the H-bond partners with those in the original crystal structure. Thirteen water molecules could not be attributed to exchangeable water molecules or less exchangeable water molecules (Table S4). E65/E312 Channel. In MD simulations using the waterremoved PSII structure, the inner space of the E65/E312 channel was entirely filled with water molecules; this suggests that almost all water molecules identified in the E65/E312 channel of the PSII crystal structure likely originate from the bulk region (Figure 2). At the inner end point of the E65/E312 channel, redox-active D1-Tyr161 (TyrZ) exists (Figure 3). The PSII crystal structure shows that a diamond-shaped cluster of water molecules21 comprised of four water molecules, including a water molecule ligated to Ca (W314), is present near TyrZ. One of the water molecules, W542 (W7 in ref 14), forms an H-bond with the phenolic O atom (Figure 3a). QM/MM studies demonstrated that the diamond-shaped cluster donates an H-bond to the phenolic O of TyrZ and effectively decreases pKa, resulting in matching pKa values for TyrZ and D1-His190; this leads to formation of the notably short, single-well H-bond between TyrZ and D1-His190 (OTyrZ−ND1‑His190 bond length, 2.46 Å14).21 Remarkably, formation of the diamond-shaped cluster of water molecules was reproduced by MD simulations starting from the water-removed PSII structure, involving W3, W358, W428, and W542 as identified in the PSII crystal structure (Figure 3). MD simulations also suggested that the water molecules that form the diamond-shaped cluster approached via the E65/E312 channel from the bulk region. The PSII crystal structure shows the low B factors of these water molecules in the PSII crystal structure (Figure 3a), which

Figure 2. Channel spaces that proceed from the Mn4CaO5 cluster (a) in the presence of the PSII protein environment and (b) in the absence of the PSII protein environment. Red dots indicate distributions of water molecules generated by the MD simulations over 5.0 ns (at 45−50 ns, 100 snapshots), starting from the waterremoved PSII structure. Red spheres indicate the crystal water molecules whose positions were reproduced (i.e., exchangeable water molecules), whereas cyan spheres indicate the crystal water molecules whose positions were not reproduced in MD simulations (i.e., less exchangeable water molecules). Dotted blue lines indicate H-bonds that form the diamond-shaped cluster of water molecules near TyrZ.21

suggests that the H-bond network of the diamond-shaped cluster near TyrZ is stable. If these water molecules were highly mobile and disordered, W542 (W714) could not provide a stable H-bond to the phenolic TyrZ. These results confirm that the diamond-shaped cluster of water molecules is a fixed dipole and can also exist in MD simulations at 300 K. The stable Hbond network is advantageous for effectively decreasing the pKa of TyrZ to form the short single-well H-bond with D1His190.21 O1−Water Chain and Cl−2 Cavity. The O1−water chain, a chain of water molecules that proceeds from the O1 site of the Mn4CaO5 cluster, is linked to the channel that is initiated near the CP43-Glu413···PsbV-Lys47 salt bridge and proceeds toward the bulk surface (O1−PsbU/V channel).17 The channel space of the O1−water chain and the connecting O1−PsbU/V channel was filled with water molecules in MD simulations starting from the water-removed PSII structure (Figure 2); this suggests that the water molecules identified in this region of the PSII crystal structure are exchangeable water molecules, arriving from the bulk region after PSII assembly. The high water accessibility can also be explained by the large channel radii and the short channel length of the O1−water chain (and O1− PsbU/V channel) with respect to the O4−water chain.17 D

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Figure 4. Penetration of water molecules from the O1−water chain into the Cl−2 cavity. (a) Pathway that connects the O1−water chain with the Cl−2 cavity, which is opened by reorientation of the D1Leu352 side chain. The dotted thick arrow indicates the movement of water molecules from the O1−water chain to the W539 binding site near the O4 site. Dotted blue lines indicate the hexagonal Cl−2−water network. Dotted circles indicate the distribution of water molecules that appear upon reorientation of the D2-Leu352 side chain. Red dots indicate distributions of water molecules generated by the MD simulations over 5.0 ns (at 45−50 ns, 100 snapshots), starting from the water-removed PSII structure. (b) Example of ∼1 Å movement of Cγ of the D2-Leu352 side chain observed in MD simulations (green) with respect to the PSII crystal structure (cyan).

Figure 3. H-Bond network of the water molecules in the E65/E312 channel. (a) B factor values of water molecules [Å2]14 are shown in blue. (b) Positions of water molecules near the TyrZ/D1-His190 moiety, obtained by MD simulations (red dots). Red dots indicate distributions of water molecules generated by the MD simulations over 5.0 ns (at 45−50 ns, 100 snapshots), starting from the water-removed PSII structure. Purple spheres indicate the ligand water molecules of the Mn4CaO5 cluster (W1−W414), which were treated as components of the Mn4CaO5 cluster in this study. Dotted blue lines indicate Hbonds that form the diamond-shaped cluster of water molecules near TyrZ.21

In the PSII crystal structure, Cl−2 and five water molecules form a hexagonal network. The hexagonal Cl−2−water network is located near the Mn4CaO5 cluster (OW548−Mn2 bond length, 3.92 Å14) but is seemingly isolated from water channels, e.g., the O1−water chain. Nevertheless, MD simulations using the water-removed PSII structure resulted in formation of a hexagonal Cl−2−water network (Figure 4a). Intriguingly, MD simulations indicated that the five water molecules that form the hexagonal Cl−2−water network came from the O1−water chain; a water molecule at the W610 binding site in the O1− water chain can enter the Cl−2 cavity via the D2-Leu352 moiety, reorienting the D2-Leu352 side chain (∼1 Å movement of Cγ) as a result of fluctuation (Figure 4b). Fluctuation of the D2-Leu352 side chain is plausible because D2-Leu352 is the carboxy-terminal residue of the D2 protein. These results identified the origin of water molecules forming the hexagonal

Cl−2−water network; D2-Leu352 could be reoriented and provide a path that connects the O1−water chain with the Cl−2 cavity. Water Chain near D1-Asn298. Near the O1−water chain, there exists a chain of water molecules that proceeds from D1Asn298 to PsbV. The water chain was proposed to be a possible proton transfer pathway from TyrZ,3,14,25 although it is not directly H-bonded to the phenolic O of TyrZ or Nε/Nδ of D1His190. In MD simulations starting from the water-removed PSII structure, the water binding sites in the H-bond network near D1-Asn298 remained unoccupied, suggesting that water molecules identified in this region of the PSII crystal structure cannot be easily exchanged with water molecules in the bulk region (Figures 1 and 2). Because no significant side chain displacement was observed near D1-Asn298 (Figure 1c), it E

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Figure 5. O4−water chain. (a) Overview. Dotted lines indicate H-bonds or ionic interactions. (b) H-Bond network of the O4−water chain with distances [Å].14

enters the E65/E312 channel from the protein bulk surface, transiently forms an H-bond with D1-Asp61, penetrates the O4−water chain, binds to the W539 site, and finally forms an H-bond with the O4 site of the Mn4CaO5 cluster. The coexistence of exchangeable (i.e., W538 and W539) and less exchangeable water molecules (i.e., W393, W397, W399, W477, W545, and W1047) in the same O4−water chain is remarkable. It was proposed that some exchangeable water molecules near the Mn4CaO5 cluster might not be visible in the PSII crystal structures because of the high level of disorder.52 However, the presence of a water molecule at W539 near the O4 site of the Mn4CaO5 cluster was reproduced in MD simulations starting from the water-removed PSII. Notably, the water molecule at W539 did not fluctuate in MD simulations using the (water-unremoved) PSII crystal structure17 because W539 interacts strongly with partners, e.g., D1-Asp61 (Figure 5b). This is consistent with the low B factor of 23.9 Å2 for W539 in the PSII crystal structure.14 Only when the W539 site was unoccupied (e.g., when W539 was moved to the catalytic site or consumed in the reaction) did a new water molecule become exergonically incorporated into the W539 site to produce H-bonds. The high binding affinity of the W539 site may lead to a decrease in the exchange rate of a water molecule at W539. The W539 site is located at the dead end of the narrow, branched E65/E312 channel, terminated by the presence of the O4 site of the Mn4CaO5 cluster, which may also contribute to a decrease in the exchange rate of a water molecule at W539. It seems likely that exchangeable water molecules near the Mn4CaO5 cluster are not necessarily disordered (or not necessarily invisible52 in the PSII crystal structure) once they bind to the binding site.

seems plausible that these water molecules are less exchangeable and seemingly tightly H-bonded, which is consistent with the proposed role of the water chain serving as a proton transfer pathway.3,14,25 These water molecules are more likely to be involved in the assembly process of the PSII complex. O4−Water Chain, Linked with the E65/E312 Channel. The O4−water chain in the D1 and CP43 proteins (Figure 5a), a chain of water molecules that are directly H-bonded to O4 of the Mn4CaO5 cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4−PsbU channel) (Figure 6).17 In MD simulations using the water-removed PSII structure, the O4− PsbU channel was filled with water molecules (Figure 6b). In contrast, the main region of the O4−water chain, where W393, W397, W399, W477, W545, and W1047 are located (Figure 5), remained unoccupied by water molecules (Figure 6c). The absence of water molecules entering the main region of the O4−water chain is explained by the narrow pore with a channel radius of less than ∼1.4 Å.17 Water molecules seem to be included in this region during the assembly of the PSII complex. Intriguingly, among all water binding sites in the O4−water chain, only the W538 and W539 sites were exceptionally occupied by water molecules in 50 ns MD simulations, despite being the most distant sites from the protein bulk surface (Figure 6c). The PSII crystal structure shows that W539 forms a notably short H-bond with the O4 site of the Mn4CaO5 cluster [OW539−O4 bond length, 2.50 Å14 or 2.32 Å19 (Figure 5)]. W539 can also form an H-bond with D1-Asp61 (OW539− OD1‑Asp61 bond length, 2.72 Å14), which is also involved in the E65/E312 channel (Figure 6c). MD simulations suggest that a water molecule near the W507/Cl−1 moiety, which originally F

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E65/E312 channel also delivered a water molecule to the W539 binding site near O4 of the Mn4CaO5 cluster (Figure 5b). From these observations, it seems possible that the E65/E312 channel, rather than the O1−water chain, plays a major role in distributing water molecules around the Mn4CaO5 cluster, possibly including substrate water molecules. On the basis of the architecture of water channels that connect the protein bulk surface with the protein interior, the inner end points may play a significant role in the protein function, utilizing water molecules. Notably, as demonstrated in the study presented here, the E65/E312 channel delivered a number of key water molecules near the Mn4CaO5 cluster (Figure 7), which implies that the E65/E312 channel is a candidate for the intake of substrate water molecules.

Figure 6. Distribution of waters along the O4−water chain and the connecting O4−PsbU channel. (a) Overview. Red dots indicate distributions of water molecules generated by the MD simulations over 5.0 ns (at 45−50 ns, 100 snapshots), starting from the water-removed PSII structure. D1-Asn338 and CP43-Pro334 form the narrowest inner space in the O4−water chain and the O4−PsbU channel. (b) Distribution of waters along the O4−PsbU channel. Red spheres indicate the crystal water molecules whose positions were reproduced (i.e., exchangeable water molecules), whereas cyan spheres indicate the crystal water molecules whose positions were not reproduced in MD simulations (i.e., less exchangeable water molecules). (c) Distributions of waters along the O4−water chain. The thick arrow indicates penetration of water molecules in the E65/E312 channel into the two water binding sites, E538 and W539, in the O4−water chain. Dotted blue lines indicate H-bond networks near TyrZ and near Cl−2.

Figure 7. Origins of the water molecules near the Mn4CaO5 cluster identified in the PSII crystal structure. Red arrows indicate penetration of water molecules into the diamond-shaped cluster near TyrZ in the E65/E312 channel and the W538 and W539 sites in the O4−water chain. Blue arrows indicate penetration of water molecules into the O1−water chain and the hexagonal Cl−2/water network. Blue dotted lines indicate key H-bond networks.

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CONCLUSIONS MD simulations performed using the water-removed PSII structure revealed the origins of key water molecules near the Mn4CaO5 cluster of the PSII crystal structure. Formation of the diamond-shaped cluster of water molecules near TyrZ, which is responsible for formation of the notably short, single-well Hbond with D1-His190,21 was reproduced by water molecules approaching along the D1-E65/D2-E312 channel. The hexagonal Cl−2/water network was reproduced by water molecules, which was penetrated from the O1 water chain with reorientation of the D2-Leu352 side chain, the carboxy terminus of the D2 protein. The crystal water at W539, which forms a particularly short H-bond with the O4 site of the Mn4CaO5 cluster in the PSII crystal structure, moved from the protein bulk surface along the D1-E65/D2-E312 channel and penetrated the O4−water chain via D1-Asp61. These findings would not have been possible by viewing the PSII crystal structure, without performing the MD simulations. In the study presented here, ligand water molecules W1−W4 were not treated as bulk water molecules but were fixed to the ligation sites, Mn4 and Ca, to appropriately represent the atomic partial charges of the entire Mn4CaO5 cluster. Fixation of the water ligands to the ligation sites would reduce the channel width near the Mn4CaO5 cluster. Nevertheless, the diamond-shaped cluster of water molecules was formed by water molecules, approaching from the protein bulk surface via the W1−W3 moieties in the E65/E312 channel (Figure 3). The

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00220. Protonation states of titratable residues, atomic charges, ligation models of the Mn4CaO5 cluster, and exchangeability of water molecules in the PSII crystal structure (PDF) Force field parameters (ZIP)

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AUTHOR INFORMATION

Corresponding Author

*Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 1538904, Japan. Telephone: +81-3-5452-5056. Fax: +81-3-54525083. E-mail: [email protected]. ORCID

Hiroshi C. Watanabe: 0000-0003-4379-8633 Hiroshi Ishikita: 0000-0002-5849-8150 G

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Biochemistry Funding

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This research was supported by JST CREST (JPMJCR1656), JSPS KAKENHI (JP17K15101 to H.C.W., JP26105012 to H.I., and JP22740276 to K.S. and H.I.), Japan Agency for Medical Research and Development (AMED), the Materials Integration for engineering polymers of Cross-ministerial Strategic Innovation Promotion Program (SIP), and the Interdisciplinary Computational Science Program in CCS, University of Tsukuba. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Theoretical calculations were partly performed at the Research Center for Computational Science (Okazaki, Japan).

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DOI: 10.1021/acs.biochem.7b00220 Biochemistry XXXX, XXX, XXX−XXX